The key to effective treatment of sepsis is rapid identification and antibiotic susceptibility testing of bloodstream pathogens. Our laboratory has developed an innovative electrochemical sensor assay for rapid (d30 min) genotypic identification of bacterial pathogens, which has been validated independently. The electrochemical sensor assay involves sandwich hybridization of bacterial rRNA to species-specific capture and detector probes. Capture probes are used to anchor target rRNA molecules to the surface of electrochemical sensors, while the detector probes generate the electrochemical signal through their link to horseradish peroxidase. Our sensor assay can be performed at room temperature and has the sensitivity to detect as few as 100 bacteria/ml. Proof of concept has been demonstrated in a clinical study of urine specimens from patients with urinary tract infection. Recently, we demonstrated that the electrochemical sensor assay can be employed to rapidly determine antimicrobial susceptibility through measurement of precursor rRNA (pre-rRNA). Because pre-rRNA is a labile, intermediate stage in the formation of mature rRNA, pre-rRNA levels respond rapidly to growth inhibition by antibiotics. Mature rRNA levels fall relatively slowly in response to antibiotics, but pre-rRNA levels drop by >10-fold within 15 min after exposure of susceptible E. coli to ciprofloxacin. More recently, we have found similar responses of bacteria to beta-lactam antibiotics. We believe that this approach can provide actionable information about bloodstream pathogens in the time frame needed to make informed decisions about antibiotic therapy. The goal of this research application is to develop a Rapid Bacterial Identification and Susceptibility (RBIS) Testing System with a time from acquisition of a positive blood culture to bacterial identification of d30 minutes and antimicrobia susceptibility testing results in d60 minutes. The Research Plan has two Specific Aims. In Specific Aim 1 we will determine whether early changes in pre-rRNA levels accurately predict antibiotic susceptibility. In Specific Aim 2 we will perform a clinical study of blinded positive blood cultures to determine the accuracy of our approach. These studies are required to generate preliminary data for a subsequent clinical trial to measure the impact of RBIS Testing System on clinical outcomes and patient care costs.